Characterization of endocytic vesicles using magnetic microbeads coated with signalling ligands.

Iron microbeads coated with the protein ligands insulin and EGF (Fe-INS and Fe-EGF) were prepared. Examination of the traffic of these ligand-coated microbeads demonstrated their internalization via clathrin-coated vesicles. Using magnetic methods, we have purified vesicles derived from the endocytic pathway. Vesicles prepared by this method are essentially free of contamination with other endomembrane compartments. Examination of the vesicles derived from cells treated with Fe-INS beads demonstrated the presence of the components of the Ras/Erk cascade on their surface. We conclude that the coupling of the Erk-signalling cascade induced by insulin takes place on the surface of endocytic vesicles derived from the internalization of the insulin receptor.